水牛TRAIL基因克隆及生物信息学分析

Cloning and Bioinformatics Analysis of TRAIL Gene in Buffalo

【目的】对水牛肿瘤坏死因子(tumor necrosis factor,TNF)相关凋亡诱导配体(TNF-related apotosis-inducing ligand,TRAIL)基因CDS序列进行克隆及序列分析,并对其编码的蛋白进行生物信息学分析,为后期TRAIL蛋白调控水牛卵巢卵泡发育、颗粒细胞增殖及凋亡的研究奠定基础。【方法】利用RT-PCR方法克隆水牛TRAIL基因CDS序列,对所获序列进行核苷酸序列、氨基酸序列相似性比对,构建系统进化树,并通过生物信息学软件分析TRAIL基因编码蛋白的结构和功能。【结果】试验成功克隆水牛TRAIL基因CDS序列,长864 bp,编码287个氨基酸;水牛TRAIL基因与牦牛、普通牛、山羊、绵羊、野猪、马、人、黑猩猩和家鼠的核苷酸序列相似性分别为99.2%、99.3%、95.9%、96.3%、84.7%、84.8%、81.3%、81.3%和70.0%。系统进化树结果表明,水牛与牦牛、普通牛的亲缘关系最近,与家鼠亲缘关系最远。氨基酸序列比对结果表明,在不同物种间,其跨膜结构域和TNF结构域序列保守性较高。TRAIL蛋白属于亲水性蛋白,存在1个跨膜结构域,140―285位氨基酸处为TNF区,具有29个磷酸化位点,无信号肽和糖基化位点,主要定位于细胞质中。TRAIL蛋白二级结构主要以无规则卷曲为主,约占51.57%,其次为延伸链(24.39%)和α-螺旋(24.04%)。TRAIL蛋白三级结构与二级结构一致,且与模型蛋白人TRAIL蛋白的相似性为75.53%。【结论】本试验克隆得到水牛TRAIL基因CDS区序列,大小为864 bp,编码287个氨基酸,水牛与牦牛、普通牛亲缘关系最近,TRAIL蛋白跨膜结构域和TNF结构域在不同物种间序列保守性较高,这可能与其功能有关。

【Objective】 The purpose of this study was to clone and analyze the CDS sequence of tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) gene in buffalo, and its coding protein was analyzed by bioinformatics, so as to prepare for the study of effects of TRAIL protein on the development of follicle, proliferation and apoptosis of granulosa cells in buffalo ovary.【Method】 The CDS sequence of TRAIL gene in buffalo was cloned by RT-PCR.The similarity of nucleotide and amino acid sequences of TRAIL gene were analyzed, the phylogenetic tree was constructed, and the structure and function of TRAIL protein were analyzed by bioinformatics softwares.【Result】 The CDS sequence of TRAIL gene in buffalo was successfully cloned, with a length of 864 bp, encoding 287 amino acids.The similarity of TRAIL gene between buffalo and Bos mutus,Bos taurus,Sus scrofa, Capra hircus, Ovis aries,Equus caballus,Pan paniscus, Homo spiens,Mus musculus were 99.2%,99.3%,95.9%,96.3%,84.7%,84.8%,81.3%,81.3% and 70.0%,respectively.The phylogenetic tree results showed that buffalo had the closest genetic relationship with Bos mutus and Bos taurus,and the farthest genetic relationship with Mus musculus.Amino acid sequence alignment results showed that the transmembrane domain and TNF domain were highly conserved among different species.TRAIL protein was a hydrophilic protein and had a transmembrane domain, the 140-285 amino acids were TNF region.There was no glycosylation site and signal peptide of TRAIL protein, which were mainly located in the cytoplasm, and had 29 phosphorylation sites.The secondary structure of TRAIL protein was mainly random coil, accounting for about 51.57%,followed by extended chain(24.39%) and alpha helix(24.04%).The tertiary structure of TRAIL protein was consistent with the secondary structure, and the similarity with the model protein TRAIL protein in human was 75.53%.【Conclusion】 In this experiment, the CDS sequence of TRAIL gene was cloned in buffalo, with a size of 864 bp and encoding 287 amino acids.The buffalo had the closest relationship with Bos mutus and Bos taurus,and the TRAIL protein transmembrane domain and TNF domain were highly conserved among different species, which might be related to its function.